The binding of insulin to its specific cell surface receptor is followed by internalization of insulin and the insulin receptor. The metabolic purposes of this process are incompletely understood. Suggested purposes for endocytosis include regulation of cell surface receptor number and termination of insulin action. Additionally, the presence of intracellular binding sites for insulin suggests that some aspects of insulin action could be produced by the interaction of internalized insulin with these sites. These issues could be clarified by studying the effects of agents which selectively inhibit insulin internalization. The Preliminary Studies section will show data identifying and characterizing a class of agents, chymotrypsin substrate analogues, which inhibit the internalization of insulin-receptor complexes in rate adipocytes. The overall goal of this application is to use these agents to characterize the metabolic consequences of inhibiting internalization in isolated rat adipocytes and cultured hepatoma cells. This goal will be achieved by determining the effects of chymotrypsin substrate analogues on glucose transport and insulin degradation in isolated rat adipocytes. The role of endocytosis in adipocyte receptor regulation will be assessed by attempting to block insulin- induced receptor loss (downregulation) with chymotrypsin substrate analogues. Cultured H-35 hepatoma cells will be employed to study effects of chymotrypsin substrate analogues on insulin stimulated glycogen synthetase, tyrosine aminotranferase and DNA synthesis. In addition, the cellular site of action of the chymotrypsin substrate analogues will be determined by attempting to identify an endogenous adipocyte chymotrypsin-like protease. Since defects in receptor regulation and insulin action are found in insulin resistant disease states, such as type II diabetes, the goals of this application are relevant to human disease. Characterizing the functional significance of insulin internalization, an early post-binding event, could eventually increase our understanding of the pathophysiology of type II diabetes mellitus.